Identification and antibiotic susceptibility of bacterial isolates from probiotic products
Introduction
The past 5 years have witnessed a strong expansion of the probiotic market and, in parallel, a rise in the number of research projects addressing fundamental and applied aspects of probiotics. New research technologies have supported earlier suggestions of health promoting properties of probiotic lactic acid bacteria (LAB) as reviewed by Naidu et al. (1999) including stabilisation of the intestinal microflora by competition against pathogens (Gibson et al., 1997), reduction of lactose intolerance (de Vrese et al., 2001), prevention of antibiotic-induced diarrhea (Pochapin, 2000), prevention of colon cancer (Wollowski et al., 2001), and stimulation of the immune system (Isolauri et al., 2001). Bringing a probiotic to the market involves a step-wise process that needs to be carefully monitored in order to obtain a correctly labeled, functional, and safe product Sanders and Huis in't Veld, 1999, Saarela et al., 2000. If a product is not labeled correctly, safety and functionality cannot be guaranteed due to lack of documentation of the product components. However, as many manufacturers rely on the widely acknowledged but occasionally debated GRAS (‘generally regarded as safe’) status of lactobacilli and bifidobacteria (Salminen et al., 1998), characterization of probiotic LAB strains with regard to taxonomic status, antibiotic resistance, and virulence may sometimes be neglected.
Microbial analyses of probiotic dairy products have demonstrated that the identity and the number of recovered species do not always correspond to the information stated on the product label Reuter, 1997, Holzapfel et al., 1998, Hamilton-Miller et al., 1999. However, it should be noted, that each of the cited studies was rather limited in number and type of products or was mainly restricted to national products. Various opinions exist as to whether it might be desirable that some probiotic strains show resistance to specific antibiotics that are, for instance, involved in antibiotic-induced diarrhea (Charteris et al., 1998). On the other hand, the commercial introduction of probiotics containing antibiotic resistant strains may also have negative consequences, for example, when resistance is transferred to intestinal pathogens (Curragh and Collins, 1992).
In the current paper, an extensive study is presented to verify the label correctness of a range of European probiotic food supplements and dairy products, together with the antibiotic susceptibility testing of the product isolates. For each of these products, the label information was checked through taxonomic characterisation of the recoverable bacterial strains using whole-cell protein profiling. In addition, individual susceptibilities were determined for a selection of six antimicrobial agents.
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Bacterial isolation
A total of 55 probiotic products, collected in eight European countries, comprised 30 dried food supplements (Table 1) and 25 dairy products (Table 2). Dairy products were collected using a refrigerated box. None of the 55 products had exceeded their expiry date. All products were examined using a set of four isolation media under standardized cultivation conditions. For the isolation of Lactobacillus and Lactococcus strains, De Man Rogosa and Sharpe Agar (MRSA) medium (CM361, Oxoid,
Bacterial isolation from probiotic products
Depending on the medium used, colony counts of the 25 investigated dairy products ranged from 105 to 109 CFU/ml. Among the 30 food supplements tested in this study, counts varied from below 1 to 106 CFU/g. During a first isolation round, we were unable to isolate viable bacteria out of 12 (i.e. 40%) of the food supplements. These products were subjected to a second isolation round including an enrichment step in MRS broth and applying anaerobic as well as micro-aerophilic conditions. Only 1 of
Discussion
Considering the significant rise in the annual consumption of probiotic products worldwide, it is important that such products are correctly labeled and that the probiotic strains are well-documented regarding safety and functionality (Sanders and Huis in't Veld, 1999). Hitherto, in Europe, there are no widely acknowledged regulations concerning the labeling issues and claims that can be made by the manufacturers of functional foods Berner and O'Donnell, 1998, Przyrembel, 2001. Our findings
Acknowledgments
This research was financially supported by a PhD grant from the Flemish Institute for the Promotion of Innovation by Science and Technology (IWT—Vlaanderen, Brussels, Belgium). Part of this research was supported by Research Programme 3G030901 of the Fund for Scientific Research—Flanders (Belgium) (F.W.O.—Vlaanderen). G.H. is a postdoctoral fellow of the Fund for Scientific Research—Flanders (Belgium) (F.W.O.—Vlaanderen). Also, we would like to thank Dirk Dewettinck, Dirk Gevers and Geert Kindt
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